Bavachin (Corylifolin) is a phytoestrogen that activates the estrogen receptors ERα and ERβ.

ERα:320nM (EC50), ERβ:680 nM (EC50)

Bavachin显著抑制黑色素合成和TYR活性。在10 μM浓度下，Bavachin抑制A375细胞中TYR和JNK蛋白的表达，以及TYR, TRP-1, TRP-2, ERK1, ERK2和JNK2 mRNA的表达。ICI182780和U0126能显著逆转Bavachin对这些蛋白和mRNA表达水平的影响[1]。在ORO染色实验中，Bavachin以剂量依赖的方式积累脂质。与对照细胞相比，10 μM Bavachin显著增加前脂肪细胞的生长，这一结果通过MTT实验确认。Bavachin还增加了前脂肪细胞增殖过程中新合成DNA中BrdU的掺入，胰岛素可以增强BrdU的掺入，而2和10 μM Bavachin与胰岛素共处理效果更佳。Bavachin激活脂肪生成因子并增加分化脂肪细胞中PPARγ的转录活性。通过Akt和AMPK途径，Bavachin增强胰岛素刺激的GLUT4介导的葡萄糖摄取[2]。BVN显著增加hMAO-A和hMAO-B活性[3]。Bavachin在与重组ER的[3H] E2竞争位点结合实验中表现出ER配体结合活性。通过使用ERα或ERβ和雌激素反应性荧光素酶质粒在CV-1细胞中的瞬时转染体系，测定了Bavachin的雌激素活性，分别为EC50 320 nM和680 nM。Bavachin增加雌激素反应性基因如pS2和PR的mRNA水平，并通过蛋白酶体途径降低ERα的蛋白水平[4]。

The chemiluminescent assay is used to confirm PCSEE MAO-A and MAO-B inhibitory effects and to test BNN and BVN hMAO-A and hMAO-B inhibition using MAO-Glo kit. Each enzyme's Arbitrary Light Unit (ALU) is measured in the presence of PCSEE, BNN, BVN, and standard DEP as an MAO-BI positive control. Briefly, hMAO-A and hMAO-B isozymes are diluted to 2× with reaction buffer (pH 7.4) and preincubated with 4× PCSEE, BNN, BVN, or DEP working solutions at RT for 30?min in white opaque 96-well plates. For determining activity inhibition, final 8.5?μg/mL concentrations of PCSEE, BNN, BVN, and DEP are used. For IC50 determination, 8× PCSEE and BNN working solutions are serially diluted using reaction buffers (pH 7.4) to make a 4× concentration. Ten points' range of PCSEE (1.0 to 250.0?μg/mL) and BNN (up to 400?μM (135.4?μg/mL)) final concentrations is used. Controls used are with and without ethanol. Ethanol solvent in controls is kept to a maximum final (volume) of ≤2%. Each isozyme is substituted with the reaction buffer for the blank. Based on our preliminary optimizations and Valley's method, the reaction is initiated by adding 4× luciferin derivative substrate (LDS) for a final (concentration) of 40 and 4?μM for hMAO-A and hMAO-B reactions, respectively. The final volume per well of each reaction is 50?μL. The reaction is optimized for the amount of A and B enzyme used to be incubated for less than 3.5?h at RT. To stop the reaction and produce the luminescence signal RLDR is added to all wells, 50?μL to each well, and incubated for a further 30?min.

Bavachin is dissolved in DMSO. MTT solution (20 μL) is added to each well of the 96-well plates, the cells are cultured for 4 h, the solution is discarded, and the purple crystal is dissolved in the wells with 150 μL DMSO solution, agitated in a 37°C incubator shaker for 10 min, and the optical density (OD) is measured at 490 nm by the microplate reader.